The vanilloid receptor (TRPV1;formerly known as VR1) plays an important role in primary hyperalgesia, a component of chronic pain. TRPV1 is functionally up-regulated in patho-physiologic states such as painful diabetic neuropathy and cystopathy. Activation of TRPV1 occurs through a number of convergent signaling pathways and emerging evidence suggests that TRPV1 function is also negatively regulated by numerous mechanisms. The discovery of novel cellular inhibitors or negative modulators of TRPV1 function and their mode of action should provide additional insights into the role of TRPV1 in peripheral pain signaling and by extension, suggest novel approaches for their application to the control of primary hyperalgesia. We propose to test the hypotheses that (i) novel cellular products play an important role in the inhibition or negative modulation of TRPV1 function and (ii) that vector-based expression of these molecules can control TRPV1 signaling in vivo. To test these hypotheses, we have devised a combination of HSV vector-based genetic strategies to identify these potential regulatory molecules and biochemical methods to characterize their role in the negative control of TRPV1 function. Specifically, we will attempt to identify products that (a) inhibit TRPV1 activation by capsaicin (CAP) and resiniferatoxin (RTX) and (b) interfere with TRPV1 potentiation by protein kinase C epsilon (PKCe) activated by Phorbol 12-myristate 13-acetate (PMA). TRPV1 inhibitory genes that either have been engineered or naturally occur, will be studied in some detail to determine the molecular mechanism underlying their ability to impede TRPV1-mediated calcium influx. In four specific aims we intend to (i) create model systems in which HSV TRPV1 expression vectors can be used to examine potential inhibitors of TRPV1 function or calcium overload (ii) create a library of vectors expressing cDNAs derived from rat dorsal root ganglia, (iii) characterize mechanisms by which the selected neuronal gene products inhibit or negatively modulate TRPV1 function and (iv) evaluate the analgesic effects of the engineered and novel TRPV1 inhibitors in vivo using rat models of pain where TRPV1 antagonism has been shown to reduce pain signaling. Both the model inhibitor genes and novel genes obtained by the HSV screening methods will be provided to Projects 1 and 2 for evaluation of their potential analgesic effects in diabetes-related neuropathic pain and models of bladder pain.